Project Summary Ultrasound has been explored as a modality to modulate nerves and muscles back in the 1920s. A number of recent studies have demonstrated the feasibility of using ultrasound to stimulate peripheral nerves, spinal cord, and brain. Yet, it has been difficult to determine whether ultrasound stimulation is via direct modulation of the membrane voltage or via indirect synaptic or network pathways. In order to unveil the mechanisms of ultrasound modulation, we formed a team of complementary expertise (Xue Han: neuroscience and technology; Ji-Xin Cheng: imaging and opto-acoustic technology; Edward Boyden: neurotechnology). Specifically, we will deploy and integrate three novel technologies that have been established in the co-PI's labs recently. First, we will use a miniature fiber optoacoustic converter (FOC) (0.4 mm in dia.) that can be positioned inside the brain to deliver localized ultrasound with an unprecedented sub-millimeter spatial resolution. Second, we will use cutting-edge genetically encoded voltage sensors to quantify the effects of ultrasound stimulation on individual cells in the brain at a temporal resolution of 1 millisecond that is beyond commonly used Ca2+ imaging. Third, we will deploy submicron spatial resolution stimulated Raman scattering microscopy to map membrane voltage at threshold and sub-threshold level to monitor membrane response to ultrasound at different regions of a single neuron. Integrating these novel technologies with a large-scale imaging platform that allows simultaneous intracranial local drug delivery, recently developed in the Han lab, we will perform a systematic analysis of the cellular and the biophysical mechanisms of ultrasound stimulation at sub-cellular level in cultured primary neurons, and in different brain regions of awake mice. Specifically, we will (1) examine the spatial response profile of individual neurons in awake brains by FOC-based neurostimulation and large-scale Ca2+ imaging in vivo; (2) examine the temporal response profile of individual neurons in awake brains by FOC-based neurostimulation and in vivo voltage imaging with genetically encoded voltage sensors; and (3) examine the involvement of membrane deformation and mechanosensitive channel activation in ultrasound neuromodulation. Our proposed studies will deliver a systematic understanding of the spatiotemporal profiles of ultrasound neuromodulation in the brain, and identify the causal role of membrane deformation and mechanosensitive channels. These new knowledge will build a new foundation for rational design of ultrasound neuro-stimulators and for basic neuroscience research as well as treatment of neurological disorders.